Glutathione is a sulfur-containing tripeptide, which functions in cellular antioxidant defense mechanisms. It is also thought to have a regulatory effect on enzymes during oxidative stress, through the formation of mixed disulfides in the proteins. Presently, there are no sensitive assays which can detect proteins that have undergone S-glutathiolation. The is partly due to the inability to obtain sensitive antibodies to glutathione because of its ubiquitous nature. As a result, our research focuses on the development of a Western analysis method involving chemical modification of these glutathiolated proteins. The model protein being used is bovine serum albumin. Mercaptoethanol-treated albumin is modified by S-glutathiolation of cysteine residues. The extent of glutathiolation is then measured with Ellman's reagent [2,2,-dithiobis(2-nitrobenzoic acid)] (DTNB). The unreacted sulfhydryl groups are then carboxymethylated, and the protein treated with thiol transferase to release glutathione. This previously S-glutathiolated cysteine is then tagged with 5-iodoacetamidofluorescein (5-IAF). A sodium-dodecyl sulfate polyacrylamide gel electrophoresis/Western analysis allows identification of the tagged proteins. This method should allow us to further investigate protein modification and regulation by glutathione during oxidative stress.